| Fluorescence Recovery after Photobleaching (FRAP) Assay to Measure the Dynamics of Fluorescence Tagged Proteins in Endoplasmic Reticulum Membranes of Plant Cells
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Author:
Date:
2014-10-20
[Abstract] In this protocol, we used fluorescence recovery after photobleaching (FRAP) to measure the influence that some mutations and drug treatment have on mobility of a green fluorescent protein (GFP)-fused viral transmembrane protein into endoplasmic reticulum membranes (Serra-Soriano et al., 2014). The proteins of interest were transiently expressed in Nicotiana benthamiana (N. benthamiana) epidermic cells by agro-infiltration. To minimize transient overexpression artifacts, fluorescence intensity values were gathered at 36 hpi using an inverted Zeiss LSM 780 confocal microscope. Only epidermic cells showing moderated expression levels and homogenous distribution through the ER of the GFP-tagged proteins were used for further experiments. To examine the role of actin ...
[摘要] 在该协议中，我们使用光漂白（FRAP）后的荧光恢复来测量一些突变和药物治疗对于将绿色荧光蛋白（GFP） - 融合的病毒跨膜蛋白移动到内质网膜中的影响（Serra-Soriano， et al。，2014）。感兴趣的蛋白质通过农杆菌浸润在烟草（Nicotiana benthamiana）（本塞姆氏烟草）表皮细胞中瞬时表达。为了使瞬时过表达伪像最小化，使用倒置Zeiss LSM 780共聚焦显微镜在36hpi收集荧光强度值。只有表现出中等表达水平和通过GFP标记的蛋白的ER的均匀分布的表皮细胞用于进一步的实验。为了检查肌动蛋白聚合在GFP标记的蛋白质的动员中的作用，我们用拉特管素B，肌动蛋白聚合的抑制剂或用DMSO作为对照预处理组织样品。使用所产生的荧光恢复曲线来获得对应于流动级分的最大荧光回收率（MFR）的百分比和最大回收的半衰期（t 1/2）/sub>）值。
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| Subcellular Localization Experiments and FRET-FLIM Measurements in Plants
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Author:
Date:
2014-01-05
[Abstract] Determining the localization of proteins within living cells may be very essential for understanding their biological function. Usually for analysis of subcellular localization, a construct encoding the translational fusion of a cDNA of interest with a fluorescent protein (FP) is engineered, transiently expressed in plant cells and examined with confocal microscopy.
In co-localization and interaction studies, two plasmids, each encoding one of the potential interacting/binding partners tagged with an appropriate pair of fluorescence proteins (for instance CFP/YFP) are co-expressed in plant cells. If proteins co-localize in certain cellular compartments it does not necessarily mean that they bind/interact to each other, therefore an additional technique should be applied for in ...
[摘要] 确定蛋白质在活细胞内的定位可能是非常必要的了解其生物学功能。通常对于亚细胞定位的分析，编码感兴趣的cDNA与荧光蛋白（FP）的翻译融合的构建体被改造，在植物细胞中瞬时表达并用共聚焦显微镜检查。在共定位和相互作用研究，在植物细胞中共表达两种质粒，每种质粒编码用适当的荧光蛋白对（例如CFP/YFP）标记的一种潜在的相互作用/结合伴侣。如果蛋白质共定位在某些细胞区室中，其不一定意味着它们彼此结合/相互作用，因此应当应用另外的技术用于体内假定相互作用的验证，/em>荧光寿命成像（FLIM）以检测荧光共振能量转移（FRET）。该协议详细描述了用于验证细菌效应物HopQ1和14-3-3a宿主之间的相互作用的方法蛋白质，并且另外检查HopQ1中的规范14-3-3结合位点中的中心丝氨酸的必要性（Giska等人，2013）。
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